1. Field of the Invention
The present invention relates to a high efficiency method of introducing DNA into poxvirus, a method of producing libraries in poxvirus, and methods of isolating polynucleotides of interest based on cell nonviability or screening methods.
2. Background Art
Identification of Disease Genes. In the past decade it has become apparent that many diseases result from genetic alterations in signaling pathways. These include diseases related to unregulated cell proliferation such as cancers, atherosclerosis and psoriasis, as well as inflammatory conditions such as sepsis, rheumatoid arthritis and tissue rejection. The finding that these proliferative diseases are based on genetic defects is the basis of new approaches for disease management by designing drugs which modulate cell signaling. In order to develop highly specific drugs, i.e., drugs which potently interfere with uncontrolled cell proliferation but which have low toxicity or side effects, it is important to identify the genes encoding polypeptides involved in the cellular signal transduction pathways whose aberrant function may result in the loss of growth control.
Although tremendous progress in understanding relevant signal transduction pathways has been made in recent years, it is clear that many of the genes involved in the development of proliferative disorders remain to be discovered.
Toxic Sequences. Several approaches have been employed for the identification and isolation of cell proliferation genes such as oncogenes and tumor suppressor genes. Traditional approaches include detection of cytogenetic abnormalities in tumor cells, kindred analysis of familial forms of cancer, and loss of heterozygosity analysis in tumor cells. Each of these classical genetic approaches is limited in the type of gene which can be isolated or in the extensive time and labor required. A faster approach would be to identify disease genes using in vitro techniques. However, a major technical limitation to the cloning of many disease genes is their negative or toxic effect on cell proliferation when present in multiple copies, such as when carried on a vector.
One approach for identifying toxic sequences involves the selection of variants that have lost certain malignancy traits, namely “revertants.” In this method, cells transformed by a variety of oncogenes are subsequently treated with a cytotoxic agent which kills dividing cells. “Revertants” that have lost the ability to rapidly divide are thus selected. However, revertant lines typically are difficult to identify and separate from the majority of rapidly growing transformed parental cells. In addition, the method may preclude the isolation of certain classes of revertants. The selection procedure may itself induce epigenetic or cytogenetic changes, thus further complicating the identification of genes responsible for the revertant phenotype.
Zarbl et al. developed an alternative assay for the selection of revertant tumor cells (Zarbl et al., 1991, Environmental Health Perspectives 93:83–89). This selection protocol is based on the prolonged retention of a fluorescent molecule within the mitochondria of a number of transformed cells relative to non-transformed cells. However, the approach is limited to particular transformation mechanisms because the prolonged dye retention phenotype is neither essential nor sufficient for cell transformation.
Other methods used to identify cell proliferation genes involve biochemical approaches for analyzing cell cycle regulators (Serrano et al., 1993, Nature 366:704–707; Xiong et al., 1993, Nature 366:701–704), random sequencing of expressed sequence tags (ESTs) and homology comparison (Lennon et al., 1996, Genomics 33:151–152), and methods for identifying differentially expressed genes, such as differential display (Liang et al., 1995, Methods Enzymol. 254:304–321). None of these approaches, however, offers a way to directly assess gene function as a method of identifying genes of interest, especially negative regulators of proliferation. Instead, candidates are identified based on a presumed (or identifiable) biochemical function or an abnormal pattern of expression. These candidates are then tested further for involvement in cancer. Such tests include mutation detection in primary cancers or cell lines, experiments using somatic cells (for example, to determine the effect of ectopic expression), or experiments in transgenic mice or knockout mice containing inactivated genes.
A more recent method for identifying cell proliferation genes involves the isolation of variants of transformed cells to identify a cell proliferation promoting activity. See U.S. Pat. No. 5,998,136. This selection system comprises the creation of growth arrested tumor cell lines or cells which undergo apoptosis by, for example, the expression of a gene encoding a growth suppressor or apoptosis-inducing gene product under the control of an inducible promoter, and selection of revertants that allow the cells to survive. Induction of the suppressor or apoptosis-inducing product causes suppression of tumor cell growth and/or cell death. Growth-proficient revertants cells are identified by virtue of their continued proliferation.
The identification of toxic molecules such as tumor suppressor genes and other inhibitors of cell proliferation to screen for potential new drugs is difficult using current technology. For example, it would be of great value to identify dominant negative mutations of signaling molecules that might be used to inhibit the unregulated growth of transformed cells. Those negative or toxic mutations that result in inhibition of cell growth or in cell death may be masked in a library or other population of cells due to the low efficiency of transfection. Additionally, such negative or toxic mutations cannot be selected for or screened using current technology because cells expressing such variants are lost from the population of transformants. These limitations may have been addressed to a limited extent by the use of inducible promoter systems, see, for example, those described in Levinson, A. D., “Gene Expression Technology,” In D. V. Goeddel (Ed.), Methods in Enzymology, Academic Press, p. 497 (1991). However, this approach is labor-intensive, is not applicable to certain situations, and has met with varied success depending on the cell type and origin of the promoter utilized.
As alluded to above, there are methods to identify positive regulators of cell growth such as oncogenes, but approaches to isolate toxic genes such as tumor suppressor genes are limited. In addition to those described above, methods for isolating negative regulators include genetic analysis based on anti-sense RNA technologies.
Another approach is a method of selection subtraction by tagging a clone in an expression library, cloning the tagged clone into a vector, delivering the tagged clone to a target cell, and comparing tags before and after selection whereby toxic genes and the attached tags disappear. See WO 99/47643.
Yet another approach selects all transformants in a population of cells before those transformants expressing negative or toxic variants are lost from the population. See WO 97/08186. This method comprises use of a cloning vector encoding a recombinant immunoglobulin molecule (rAb) that is specific for a particular hapten and expressed on the cell surface. Cells receiving the vector express the rAb early after transfection, and are separated from the non-recipient cells by the ability to bind the cognate hapten conjugated to a solid surface, such as beads. This method does not distinguish recipients expressing a gene or cDNA of interest, e.g., a negative or toxic variant, from the remaining recipients.
Differentially Expressed Sequences. Cloning, sequencing, and identification of function of mammalian genes is a first priority in a genomic based drug discovery. In particular, it is important to identify and make use of genes which are spatially and/or temporally regulated in an organism, for example, genes involved in differentiation and growth regulation.
Animal model systems such as the fruit fly and the worm are often used in gene identification because of ease of manipulation of the genome and ability to screen for mutants. While these systems have their limitations, large numbers of developmental mutations have been identified in those organisms either by monitoring the phenotypic effects of mutations or by screening for expression of reporter genes incorporated into developmentally regulated genes.
Many features of the mouse make it the best animal model system to study gene function. However, the mouse has not been used for large scale classical genetic mutational analysis because random mutational screening and analysis is very cumbersome and expensive due to long generation times and maintenance costs.
A disadvantage in using animal models for the identification of genes is the need to establish a transgenic animal line for each mutational event. This disadvantage is alleviated in part by using embryonic stem (ES) cell lines because mutational events may be screened in vitro prior to generating an animal. ES cells are totipotent cells isolated from the inner cell mass of the blastocyst. Methods are well known for obtaining ES cells, incorporating genetic material into ES cells, and promotion of differentiation of ES cells. ES cells may be caused to differentiate in vitro or the cells may be incorporated into a developing blastocyst in which the ES cells will contribute to all differentiated tissues of the resulting animal. Vectors for transforming ES cells and suitable genes for use as reporters and selectors are also well known.
Gene entrapment strategies also have been employed to identify developmentally regulated genes. One type of entrapment vector is called a “promoter trap,” which consists of a reporter gene sequence lacking a promoter. Its integration is detected when the reporter is integrated “in-frame” into an exon. In contrast, a “gene trap” vector targets the more prevalent introns of the eucaryotic genome. The latter vector consists of a splice-acceptor site upstream from a reporter gene. Integration of the reporter into an intron results in a fusion transcript containing RNA from the endogenous gene and from the reporter gene sequence.
Gene trap vectors may be made more efficient by incorporation of an internal ribosomal entry site (IRES) such as that derived from the 5′ non-translated region of encephalomyocarditis virus (EMCV). Placement of a IRES site between the splice acceptor and the reporter gene of a gene trap vector means the reporter gene product need not be translated as a fusion product with the endogenous gene product, thereby increasing the likelihood that integration of the vector will result in expression of the reporter gene product.
Gossler, A., et al. Science 244:463–465 (1989) describe the use of enhancer trap gene trap vectors for use in identifying developmentally regulated genes. The gene trap vector consists of the mouse En-2 splice acceptor upstream from lacZ (reporter) and a selector gene (hBa-neo). This and other current methods requires elaborate screening procedures for linking a mutation to a particular spacial/temporal scheme or event whereby the mutation is detected in the relevant tissue.
A more recently developed method is complementation trapping. See WO 99/02719. This method makes use of known genes whose expression is restricted to specific tissue, tissues or specialized cells (“restricted expression”) to facilitate identification and manipulation of new genes and their associated transcription control elements which have similar patterns of expression. The method comprises (i) transforming a eucaryotic cell with a DNA sequence encoding a first indicator component under the control of a promoter having restricted expression; (ii) transforming the cell of (i) or a descendent of the cell of step (i), by operably integrating into the cell's genome DNA lacking a promoter but which comprises a sequence encoding a second indicator component; (iii) producing tissue or specialized cells from the cell of (ii); and (iv) monitoring the tissue or specialized cells of (iii) for a detectable indicator resulting from both the first and second indicator components.
Expression Libraries. A basic tool in the field of recombinant genetics is the conversion of poly(A)+ mRNA to double-stranded (ds) cDNA, which then can be inserted into a cloning vector and expressed in an appropriate host cell. A substantial number of variables affect the successful cloning of a gene of interest and cDNA cloning strategy thus must be chosen with care. A method common to many cDNA cloning strategies involves the construction of a “cDNA library” which is a collection of cDNA clones derived from the poly(A)+ mRNA derived from a cell of the organism of interest.
A mammalian cell may contain up to 30,000 different mRNA sequences, and the number of clones required to obtain low-abundance mRNAs, for example, may be much greater. Methods of constructing genomic eukaryotic DNA libraries in different expression vectors, including bacteriophage lambda, cosmids, and viral vectors, are known. Some commonly used methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, N.Y. (1982).
Once a genomic cDNA library has been constructed and expressed in host cells, it is necessary to isolate from the thousands of host cells the particular cell or cells which contain the particular gene of interest. Many different methods of isolating target genes from cDNA libraries have been utilized, with varying success. These include, for example, the use of nucleic acid probes, which are labeled mRNA fragments having nucleic acid sequences complementary to the DNA sequence of the target gene. When this method is applied to cDNA clones of abundant mRNAs in transformed bacterial hosts, colonies hybridizing strongly to the probe are likely to contain the target DNA sequences. The identity of the clone then may be proven, for example, by in situ hybridization/selection (Goldberg et al., Methods Enzymol., 68:206 (1979)) hybrid-arrested translation (Paterson et al., Proceedings of the National Academy of Sciences, 74:4370 (1977)), or direct DNA sequencing (Maxam and Gilbert, Proceedings of the National Academy of Sciences, 74:560 (1977); Maat and Smith, Nucleic Acids Res., 5:4537 (1978)).
Such methods, however, have major drawbacks when the object is to clone mRNAs of relatively low abundance from cDNA libraries. For example, using direct in situ colony hybridization, it is very difficult to detect clones containing cDNA complementary to mRNA species present in the initial library population at less than one part in 200. As a result, various methods for enriching mRNA in the total population (e.g. size fractionation, use of synthetic oligodeoxynucleotides, differential hybridization, or immunopurification) have been developed and are often used when low abundance mRNAs are cloned. Such methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra.
Use of mammalian expression libraries to isolate cDNAs encoding mammalian proteins such as those described above would offer several advantages. For example, the protein expressed in a mammalian host cell should be functional and should undergo any normal posttranslational modification. A protein ordinarily transported through the intracellular membrane system to the cell surface should undergo the complete transport process. A mammalian expression system also would allow the study of intracellular transport mechanisms and of the mechanism that insert and anchor cell surface proteins to membranes. Further, use of a mammalian system would make it possible to isolate polynucleotides based on functional expression of mammalian RNA or protein.
One common mammalian host cell, called a “COS” cell, is formed by infecting monkey kidney cells with a mutant viral vector, designated simian virus strain 40 (SV40), which has functional early and late genes, but lacks a functional origin of replication. In COS cells, any foreign DNA cloned on a vector containing the SV40 origin of replication will replicate because SV40 T antigen is present in COS cells. The foreign DNA will replicate transiently, independently of the cellular DNA.
With the exception of some recent lymphokine cDNAs isolated by expression in COS cells (Wong, G. G., et al., Science 228:810–815 (1985); Lee, F. et al., Proc. Natl. Acad. Sci. USA 83.2061–2065 (1986); Yokota, T., et al., Proc. Natl. Acad. Sci. USA 83:5894–5898 (1986); Yang, Y., et al., Cell 47:3–10 (1986)), however, few cDNAs in general are isolated from mammalian expression libraries. There appear to be two principal reasons for this: First, the existing technology (Okayama, H. et al., Mol. Cell. Biol. 2:161–170 (1982)) for construction of large plasmid libraries is difficult to master, and library size rarely approaches that accessible by phage cloning techniques. (Huynh, T. et al., In: DNA Cloning Vol. I, A Practical Approach, Glover, D. M. (ed.), IRL Press, Oxford (1985), pp. 49–78). Second, the existing vectors are, with one exception (Wong, G. G., et al., Science 228:810–815 (1985)), poorly adapted for high level expression, particularly in COS cells. The reported successes with lymphokine cDNAs do not imply a general fitness of the methods used, since these cDNAs are particularly easy to isolate from expression libraries: Lymphokine bioassays are very sensitive ((Wong, G. G., et al., Science 228:810–815 (1985); Lee, F. et al., Proc. Natl. Acad. Sci. USA 83:2061–2065 (1986); Yokota, T. et al., Proc. Natl. Acad. Sci. USA 83:5894–5898 (1986); Yang, Y. et al., Cell 47:3–10 (1986)) and the mRNAs are typically both abundant and short (Wong, G. G. et al., Science 228:810–815 (1985); Lee, F., et al., Proc. Natl. Acad. Sci. USA 83:2061–2065 (1986); Yokota, T., et al., Proc. Natl. Acad. Sci. USA 83:5894–5898 (1986); Yang, Y., et al., Cell 47:3–10 (1986)).
Thus, expression in mammalian hosts previously has been most frequently employed solely as a means of verifying the identity of the protein encoded by a gene isolated by more traditional cloning methods. For example, Stuve et al., J. Virol. 61(2):327–335 (1987), cloned the gene for glycoprotein gB2 of herpes simplex type II strain 333 by plaque hybridization of M13-based recombinant phage vectors used to transform competent E. coli JM101. The identity of the protein encoded by the clone thus isolated was verified by transfection of mammalian COS and Chinese hamster ovary (CHO) cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation.
Oshima et al. used plaque hybridization to screen a phage lambda gt11 cDNA library for the gene encoding human placental beta-glucuronidase. Oshima et al., Proceedings of the National Academy of Sciences, U.S.A. 84:685–689 (1987). The identity of isolated cDNA clones was verified by immunoprecipitation of the protein expressed by COS-7 cells transfected with cloned inserts using the SV40 late promoter.
Transient expression in mammalian cells has been employed as a means of confirming the identity of genes previously isolated by other screening methods. Gerald et al., Journal of General Virology 67:2695–2703(1986). Mackenzie, Journal of Biological Chemistry 261:14112–14117 (1986); Seif et al., Gene 43:1111–1121 (1986); Orkin et al., Molecular and Cellular Biology 5(4):762–767 (1985). These methods often are inefficient and tedious and require multiple rounds of screening to identify full-length or overlapping clones. Prior screening methods based upon expression of fusion proteins are inefficient and require large quantities of monoclonal antibodies. Such drawbacks are compounded by use of inefficient expression vectors, which result in protein expression levels that are inadequate to enable efficient selection.
Seed et al., U.S. Pat. No. 5,506,126 developed a cloning technique based upon transient expression of antigen in eukaryotic cells and physical selection of cells expressing the antigen by adhesion to an antibody-coated substrate, such as a culture dish. This method for cloning cDNA encoding a cell surface antigen comprises preparing a cDNA library; introducing this cDNA library into eukaryotic mammalian cells; culturing the cells under conditions allowing expression of the cell surface antigen; exposing the cells to a first antibody or antibodies directed against the cell surface antigen, thereby allowing the formation of a cell surface antigen-first antibody complex; subsequently exposing the cells to a substrate coated with a second antibody directed against the first antibody, thereby causing cells expressing the cell surface antigen to adhere to the substrate via the formation of a cell surface antigen-first antibody-second antibody complex; and separating adherent from non-adherent cells. However, this method is limited to the isolation and cloning of proteins which are expressed and transported to the cell surface, whose expression does not adversely affect cell viability, and for which specific antibody has been isolated.
Poxvirus Vectors. Poxvirus vectors are used extensively as expression vehicles for protein and antigen expression in eukaryotic cells. The ease of cloning and propagating vaccinia in a variety of host cells has led to the widespread use of poxvirus vectors for expression of foreign protein and as vaccine delivery vehicles (Moss, B. 1991, Science 252:1662–7).
Customarily, a foreign protein coding sequence is introduced into the poxvirus genome by homologous recombination. In this method, a previously isolated foreign DNA is cloned in a transfer plasmid behind a vaccinia promoter flanked by sequences homologous to a region in vaccinia which is non-essential for viral replication. The transfer plasmid is introduced into vaccinia virus-infected cells to allow the transfer plasmid and vaccinia virus genome to recombine in vivo via homologous recombination. As a result of the homologous recombination, the foreign DNA is transferred to the viral genome.
Although homologous recombination is efficient for transferring previously isolated foreign DNA of relatively small size into vaccinia virus, the method is much less efficient for transferring large inserts, for constructing libraries, and for transferring foreign DNA which is deleterious to bacteria.
Alternative methods using direct ligation vectors have been developed to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination (Merchlinsky, M. et al., 1992, Virology 190:522–526; Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA. 89:9977–9981). In such protocols, the DNA from the genome is digested, ligated to insert DNA in vitro, and transfected into cells infected with a helper virus (Merchlinsky, M. et al., 1992, Virology 190:522–526, Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA 89:9977–9981). In one protocol, the genome was digested at a unique NotI site and a DNA insert containing elements for selection or detection of the chimeric genome was ligated to the genomic arms (Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA. 89:9977–9981). This direct ligation method was described for the insertion of foreign DNA into the vaccinia virus genome (Pfleiderer et al., 1995, J. General Virology 76:2957–2962). Alternatively, the vaccinia WR genome was modified by removing the NotI site in the HindIII F fragment and reintroducing a NotI site proximal to the thymidine kinase gene such that insertion of a sequence at this locus disrupts the thymidine kinase gene, allowing isolation of chimeric genomes via use of drug selection (Merchlinsky, M. et al., 1992, Virology 190:522–526).
The direct ligation vector vNotI/tk allows one to efficiently clone and propagate previously isolated DNA inserts at least 26 kilobase pairs in length (Merchlinsky, M. et al., 1992, Virology, 190:522–526). Although large DNA fragments are efficiently cloned into the genome, proteins encoded by the DNA insert will only be expressed at the low level corresponding to the thymidine kinase gene, a relatively weakly expressed early class gene in vaccinia. In addition, the DNA will be inserted in both orientations at the NotI site.
The cloning methods and the selection methods above have a number of drawbacks and limitations. Therefore it is desirable, and the objective of the present invention, to develop cloning and selection methods that would permit the identification and isolation of novel genes based on functional analysis.